Large-scale purification of synthetic oligonucleotides and carcinogen-modified oligodeoxynucleotides on a reverse-phase polystyrene (PRP-1) column.
A procedure is described for the large-scale purification of synthetic oligonucleotides using a polystyrene (PRP-1, Hamilton Co.) high-performance liquid chromatography (HPLC) column with a phosphate/methanol/acetonitrile solvent system. Pure oligonucleotides are obtained with a three-step procedure that involves only one column purification step. The dimethoxytrityl group is left on the oligomer for the HPLC purification. The use of the PRP-1 polystyrene column with a phosphate/methanol/acetonitrile solvent system provides excellent separation of the desired dimethoxytrityl-bearing oligonucleotide from failure sequences. The dimethoxytrityl group is removed by treatment with acetic acid and the oligonucleotide is desalted on a C-18 Sep-Pak cartridge. The oligodeoxynucleotides obtained are shown to be essentially pure by HPLC, polyacrylamide gel electrophoresis, and 500-MHzNMR spectroscopy. This procedure is especially useful for the large-scale purification of oligonucleotides required for NMR studies. The PRP-1 column and the phosphate/methanol/acetonitrile solvent system is useful for purifying modified oligonucleotides containing lipophilic groups such as the carcinogen 2-(acetylamino)fluorene.
Huang
G J
GJ
Department of Chemistry, University of Rochester, New York 14627.
Krugh
T R
TR
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D005021:Ethylamines; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification* / Q000378:metabolism; D009841:Oligonucleotides / Q000302:isolation & purification* / Q000378:metabolism; D011137:Polystyrenes; D014320:Triphenylmethyl Compounds
D005021:Ethylamines; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D011137:Polystyrenes; D014320:Triphenylmethyl Compounds; D004247:DNA; C016162:triethylamine